Prevention of dental plaque is important in maintaining the health of the oral cavity. Thus, new dental care compositions are constantly sought that are more effective in preventing dental plaque buildup or aid in its removal. Additionally, once cells which cause dental plaque bind to a tooth surface to form a biofilm, dental appliances must be used to remove the cells to prevent tissue irritation that leads to gingivitis, and potentially to periodontal disease or peri-implantitis.
Bacterial adherence and colonization are considered key factors in the etiology of dental plaque and biomaterial-based infections. Beachey E. H., ed. Bacterial Adherence, Chapman and Hall, London, 1980; Gristina A. G., Science 237:1588-1595, 1987. S. mutans has been strongly implicated as the etiological agent of dental caries (Menaker L., The Biologic Basis of Dental Caries: An Oral Biology textbook. Harper & Row Publishers, Hagerstow, Md., 1980) and is involved in early dental plaque formation, as is A. naeslundii (Socransky et al., J Periodont Res 12:90-106, 1977). P. gingivalis is a periodontopathic bacterium that has been associated with periodontal disease. van Steenbergen T. J. M., van Winkelhoff A. J., de Graaff J., In Periodontal Disease: Pathogens & Host Immune Responses, Hamada S., Holt S. C. McGhee J. R., Eds., Quintessence Publishing Co. Ltd., Chicago, pp. 41-52, 1991. It is now well appreciated that dental plaque results from the binding and growth of many other bacterial species on the tooth surface and that there are complex interbacterial relationships that are involved in dental plaque formation and growth. See Tanner, et al., J. Clinical Periodontol. 25:85-98, 1998.
Removal of dental plaque from a tooth surface usually requires direct contact between an oral care product and the plaque biofilm. There are many different types of personal dental appliances that purport to remove tooth plaque. Personal dental appliances, such as a toothbrush, rely on direct contact between the toothbrush bristles and the tooth surface to remove dental plaque. Powered toothbrushes move the bristles across the tooth to remove plaque while relying on the user to position the toothbrush about the dentition. Other dental instruments such as proxibrushes, plaque removers, and floss also rely on direct contact with the plaque accumulations to clean the tooth surface. As an alternative to direct bristle contact, oral irrigators are meant to remove plaque from teeth via fluid forces. Irrigators project a high velocity fluid jet that may be directed into areas where the bristles of a toothbrush cannot reach.
Determining the efficacy of an oral care product, whether it be an oral composition for application to the oral cavity whose purpose is to prevent the formation of plaque, or a dental appliance which is designed to remove dental plaque, can be a slow and costly process. Clinical studies are particularly expensive. Therefore, a variety of alternative test methods have been devised which allow for more time effective and less expensive determination of the efficacy of an oral care product.
Viccaro (U.S. Pat. No. 4,430,320) discloses the use of a bacterial model system to test the effectiveness of ammonium fluorometallate compounds in controlling tooth decay and plaque development. The effectiveness of the ammonium fluorometallate compounds against plaque formation was determined using an in vitro plaque development assay. Aluminum plummets pre-coated with saliva were placed into bacterial growth medium with clinical plaque samples. After a first incubation period to allow bacterial attachment and growth, the plummets were treated with the test composition and further incubated overnight in a saliva solution. On the second day the plummets were retreated with the test solution and reincubated in bacterial growth medium. The amount of bacteria growing on the surface of the plummets was quantitated by first sonicating the plummets to dislodge the bacteria into a sonication solution and then placing the solution into a spectrophotometer to determine optical density at 570 nm.
Gaffar et al., (U.S. Pat. No. 5,368,845) tested antiplaque compositions in an "artificial mouth" which pumped a constant flow of human saliva through a chamber containing two germanium plates. The test composition was pumped through the mouth for a set time period, followed by pumping a mixture of saliva (containing bacteria) and a bacterial growth medium for 24 to 48 hours. Composition efficacy was determined by performing infrared spectroscopy on the germanium plates to quantitate the amount of bacteria bound to the plates.
More commonly, the effectiveness of an antiplaque agent is determined by performing serial dilution experiments of the agent into cultures of oral bacteria to calculate the minimum inhibitory concentration, or MIC, of the agent. The MIC is the minimum concentration in micrograms per milliliter of an antimicrobial agent at which no bacterial growth is observed. At concentrations below the MIC, an antimicrobial agent is ineffective at killing or inhibiting the growth and reproduction of bacteria. At concentrations above the MIC, an antimicrobial agent is effective at killing or inhibiting the growth and reproduction of bacteria. See, for example, Nabi, et al., U.S. Pat. No. 5,472,684.
The primary drawback to using the serial dilution test on bacterial cultures to calculate a MIC of an antiplaque or antimicrobial agent is that the sensitivity of a bacterium within an in vivo multispecies plaque biofilm is different as compared to a bacterium growing in a liquid monoculture. See, for example, Millward and Wilson, Microbios., 58:155-164, 1989.
Huntley, et al., (J. Dent. Res., 78:343, Abst. 1897, 1999) devised an assay to measure the efficacy with which a mouth rinse kills bacteria in a biofilm rather than a planktonic liquid suspension culture. A bacterial biofilm was formed on the bottom surface of each well in a microtiter plate. Each biofilm was exposed to a mouth rinse in a time course experiment. At each assay point bacteria in the biofilm were rinsed to remove the mouth rinse and dead bacteria, and then detached from the microtiter plate well surface by sonication. The amount of alive bacteria present in the biofilm that survived the mouth rinse treatment was measured using a bioluminescence assay that measures the amount of ATP present.
One goal in the design of new dental appliances is to devise tools that are easy to use yet are effective at removing plaque from the interproximal gap regions between adjacent teeth and the subgingival region where the tooth emerges from the gum. Current appliance test methods measure the ability of a product to remove bacteria from a test biofilm surface after direct contact between the biofilm surface and the appliance (See Wu-Yuan, et al., 1994, J. Clin. Dent., 3:89-93 (FIG. 2 and Table 1)), or by maintaining the appliance at a fixed distance from a biofilm surface (See Wu-Yuan, et al., 1994, J. Clin. Dent., 3:89-93; Stanford et al., 1997, J. Clin. Dent., 8:10-14; Smith et al, J. Dent. Res., 78:414, Abst. 2469, 1999).
Effective methods are needed that allow new dental appliances to be tested to determine the efficacy with which they remove plaque bacteria and other cells from subgingival and interproximal regions. In particular, methods are needed that provide an accurate basis for comparative studies between different types of dental appliances to determine their relative efficacies in plaque removal from tooth surfaces that are hard to brush, i.e., interproximal and subgingival surfaces. In addition, methods are needed to determine the efficacy of anti-plaque formulations at preventing or inhibiting plaque development.